Protein C in its activated form plays an important role in regulating blood coagulation. The activated protein C, a serine protease, inactivates coagulation Factors Va and VIIIa by limited proteolysis. The coagulation cascade initiated by tissue injury, for example, is prevented from proceeding in an unimpeded chain-reaction beyond the area of injury by activated protein C.
Protein C is synthesized in the liver as a single chain precursor polypeptide which is subsequently processed to a light chain of about 155 amino acids (M.sub.r =21,000) and a heavy chain of 262 amino acids (M.sub.r =40,000). The heavy and light chains circulate in the blood as a two-chain inactive protein, or zymogen, held together by a disulfide bond. When a 12 amino acid residue peptide is cleaved from the amino terminus of the heavy chain portion of the zymogen in a reaction mediated by thrombin, the protein becomes activated. The N-terminal portion of the light chain contains nine .gamma.-carboxyglutamic acid (Gla) residues that are required for the calcium-dependent membrane binding and activation of the molecule. Another blood protein, referred to as "protein S", is believed to accelerate the protein C-catalyzed proteolysis of Factor Va.
Protein C has also been implicated in the action of tissue-type plasminogen activator (Kisiel et al., Behring Inst. Mitt. 73:29-42, 1983). Infusion of bovine activated protein C (APC) into dogs results in increased plasminogen activator activity (Comp et al., J. Clin. Invest. 68:1221-1228, 1981). Other studies (Sakata et al., Proc. Natl. Acad. Sci. USA 82:1121-1125, 1985) have shown that addition of APC to cultured endothelial cells leads to a rapid, dose-dependent increase in fibrinolytic activity in the conditioned media, reflecting increases in the activity of both urokinase-related and tissue-type plasminogen activators. APC treatment also results in a dose-dependent decrease in anti-activator activity. In addition, studies with monoclonal antibodies against endogenous APC (Snow et al., FASEB Abstracts, 1988) implicate APC in maintaining patency of arteries during fibrinolysis and limiting the extent of tissue infarct.
Experimental evidence indicates that protein C may be clinically useful in the treatment of thrombosis. Several studies with baboon models of thrombosis have indicated that activated protein C in low doses will be effective in prevention of fibrin deposition, platelet deposition and loss of circulation (Gruber et al., Hemostasis and Thrombosis 374a: abstract 1512, 1988; Widrow et al., Fibrinolysis 2 suppl. 1: abstract 7, 1988; Griffin et al., Thromb. Haemostasis 62: abstract 1512, 1989).
In addition, exogenous activated protein C has been shown to prevent the coagulopathic and lethal effects of gram negative septicemia (Taylor et al., J. Clin. Invest. 19:918-925, 1987). Data obtained from studies with baboons suggest that activated protein C plays a natural role in protecting against septicemia.
Until recently, protein C was purified from clotting factor concentrates (Marlar et al., Blood 59:1067-1072, 1982) or from plasma (Kisiel, J. Clin. Invest. 64:761-769, 1979) and activated in vitro. However, the possibility that the resulting product could be contaminated with such infectious agents as hepatitis virus, cytomegalovirus, or human immunodeficiency virus (HIV) make the process unfavorable.
While expression of protein C through recombinant means has been theoretically possible as the genes for both human and bovine protein C are known (Foster et al., Proc. Natl. Acad. Sci. USA 82:4673-4677, 1985; Foster et al., Proc. Natl. Acad Sci. USA 81:4766-4770, 1984 and U.S. Pat. No. 4,775,624), it has been met with limited success. Expression of some vitamin K-dependent proteins, such as protein C in cultured cells, has not produced protein C that has been at both commercially valuable levels and biologically functional when activated (i.e. had anticoagulant activity (Grinnell et al., in Bruley and Drohn, eds., Protein C and Related Anticoagulants:29-63, Gulf Publishing, Houston, Tex. and Grinnell et al., Bio/Technol. 5:1189-1192, 1987)). Transgenic expression of protein C has yielded somewhat higher levels of expression, but the recombinant protein's anticoagulant activity has still remained low, with less than 50% of the material having biological activity (Velander et al., Proc. Natl. Acad. Sci. USA 89:12003-12007, 1992). Therefore, there remains a need for producing protein C that is both expressed at high levels and has therapeutic value.